Project Summary Immunization with antigen in the presence of agonists for both a Toll Like Receptor (TLR) and CD40 (combined TLR/CD40 immunization) elicits a vigorous expansion of antigen-specific CD8+ T cells that is exponentially greater than the response elicited by either agonist alone. Not only is the primary immune response to this vaccination robust, it also forms long lived, CD8+ T cell memory that can protect against future infectious challenge even in the absence of CD4+ T cells. This has been recently verified in non-human primates, where the vaccine produced responses exponentially stronger than responses to typical viral vectors. Given the potency and clinical potential for this vaccine adjuvant platform, it is critical that we understand its molecular and cellular mechanistic underpinnings. We made the discovery that T cell responses to adjuvanted subunit vaccinations were unexpectedly and completely dependent on the cytokines IL-27 and IL-15. This was surprising because the T cell response to an infectious challenge proceeds unabated, and can even be elevated (for IL-27 deficiency), in the absence of these cytokines. More recently, we have identified the expression of the transcription factor IRF4 as a major result of IL-27/15 signaling during vaccination. IRF4 is well documented to facilitate aerobic glycosylation as the source of energy and biomass generation for T cells during the primary immune response. However, we found that T cells responding to vaccination almost exclusively utilized mitochondrial respiration, a metabolism that is supposed to be used only by nave and memory T cells and not cells undergoing the dramatic proliferative burst of the primary response. Thus, IRF4 appears to be facilitating very different kinds of metabolic programs in a T cell depending on whether or not it has been encountered the inflammatory environment of a subunit vaccine or of an infection. These and other observations make us conclude that the rules behind robust subunit vaccine-elicited immunity appear to be substantially different than those guiding infectious responses. This proposal will use cutting edge methods and approaches to fully understand the nature of this difference and will test i) how IL-27 and IL-15 influence downstream transcription factor networks, ii) how these transcriptional elements are tied into vaccine- vs. infection-driven programs of T cell activation and expansion, and iii) how the earliest T-DC interactions might influence both transcription factor expression and downstream influence on metabolic programming of the primary CD8+ T cell response.